Anhydride enzyme intermediates

In the kinetically controlled synthesis, an activated acyl donor (ester, amide, or anhydride) is used to form an acyl-enzyme intermediate. 

One possible mechanism for the hydrolysis of peptides or esters by carboxypeptidase A involves two steps with an anhydride (acyl-enzyme) intermediate.418 In the first step, the zinc(II) activates the substrate carbonyl group towards nucleophilic attack by a glutamate residue, resulting in the production of a mixed anhydride (127). Breakdown of the anhydride intermediate is rate determining with some substrates.419 An understanding of the chemistry of metal ion effects in anhydride hydrolysis is therefore of fundamental importance in regard to the mechanism of action of the enzyme. Until recently there have been few studies of metal ion-catalysed anhydride solvolysis. 

Scheme 8 Two mechanistic proposals for the catalytic mechanism of CoA-transferases. In mechanism A, an acyl-enzyme Intermediate Is formed by reaction of an enzyme-bound glutamate (aspartate for Class III enzymes) with the donor acyl-CoA, followed by the formation of an enzyme-bound glutamyl- (or aspartyl-) CoA thioester Intermediate. The thioester subsequently reacts with the acceptor carboxylate to give a new acyl-enzyme anhydride from which the acyl group Is transferred to CoA. In Class I transferases, this process follows classical ping-pong kinetics, whereas In Class III enzymes the donor carboxylate only leaves the enzyme complex upon formation of the product (see text for details). Mechanism B represents a ternary complex mechanism as used by Class II enzymes In which a transient anhydride made up of the donor and acceptor acyl groups Is formed by reaction of the acceptor carboxylate with the donor acyl-ACP. The free ACP subsequently reacts with this anhydride to complete acyl transfer.

The role of the metal ion in ester hydrolysis catalysed by CPA has been examined with both Zn +- and Co +-substituted enzymes. When the terminal carboxyl of the substrate is electrostatically linked to argenine-145 and the aromatic side-chain lies in a hydrophobic pocket, the only residues close enough to the substrate to enter catalysis are glutamate-270, tyrosine-248, the metal ion, and its associated water. Low-temperature studies aid the elucidation of the mechanism. Between - 25 and - 45 °C in ethylene glycol-water mixtures two kinetically discrete processes are detected, the slower of which corresponds to the catalytic rate constant. The faster reaction is interpreted as deacylation of a mixed anhydride acyl-enzyme intermediate formed by nucleophilic attack by glutamate-270 on the substrate (Scheme 6). Differences in the acidity dependences of the catalytic rate constant with the metal ions Zn + (p STa 6.1) and Co +-(pATa 4.9) suggest that ionization of the metal-bound water molecule occurs and is involved in the decay of the anhydride. The catalytic rate constant shows an isotope effect in DgO. 

The mechanism shown above involves two steps and an anhydride (acyl-enzyme) intermediate. In the first step Zn(II) of the enzyme electrophically activates the substrate carbonyl towards nucleophilic attack by a glutamate residue. Departure of an alkoxyl group (with ester substrates) or an amino group (with peptide substrates) results in the production of an anhydride between the enzyme glutamate residue and the scissile carboxyl group. In the second step the hydrolysis of this anhydride can be catalyzed by the... 

Therefore, a key argument in regard to the proposed enzymatic mechanism of carboxypeptidase A is whether the carboxylate group of Glu-270 is steri-cally capable of participating efficiently in a nucleophilic reaction. In fact, such evidence has now been obtained by spectral characterization in the subzero temperature range (—60°C) study of a covalent acyl-enzyme intermediate obtained in the hydrolysis of the specific substrate 0-(trans-p-chlo-rocinnamoyl)-L-jS-phenyllactate by carboxypeptidase A (224). Furthermore, the results indicate that deacylation of the mixed anhydride intermediate is catalyzed by a Zn-bound hydroxide group. 

On the basis of exchange studies a uridyl-enzyme intermediate has been postulated in the synthesis of UDP-glucose (S41S). The same type of intermediate might be common to all of the reactions in which nucleotide-phosphoric anhydrides are formed. 

An enzymatic reaction intermediate formed by phospho-ryl transfer to a carboxyl group on an enzyme. Acyl-phosphates are structurally analogous to acid anhydrides (R—CO —O —CO—R ), and they are thermodynamically less stable than either of the two phosphoanhydride bonds in ATP. This is evident by the fact that the acetate kinase reaction (ADP + acetyl-phosphate = ATP + acetate) favors ATP formation with an equilibrium constant of about 3,000. Acetyl-phosphate can be chemically synthesized by reacting orthophosphate with acetic anhydride. 

This reaction occurs in two steps in the enzyme s active site. In step (Fig. 27-14) an enzyme-bound intermediate, aminoacyl adenylate (aminoacyl-AMP), forms when the carboxyl group of the amino acid reacts with the a-phosphoryl group of ATP to form an anhydride linkage, with displacement of pyrophosphate. In the sec-... 

Reduction -of benzene [BENZENE] (Vol 4) -dehydrogenase-catalyzed [ENZYMES IN ORGANIC SYNTHESIS] (Vol 9) -m dyes manufacture pYES AND DYE INTERMEDIATES] (Vol 8) -of esters pSTERS, ORGANIC] (Vol 9) -maleic anhydride [MALEIC ANHYDRIDE, MALEIC ACID AND FUMARIC ACID] (Vol 15) -microbial transformations [MICROBIAL TRANSFORMATIONS] (Vol 16) -of mtrophenols [AMINOPHENOLS] (Vol 2) -of terphenyls [BIPHENYL AND TERPHENYLS] (Vol 4)... 

For thiosulfate oxidation it is hypothesized that an enzyme system on the cell surface initiates the formation of polythiosulfonic acid and consequently splits the terminal S03" as sulfate. This process may evolve the intermediate of mixed anhydride -S-O-PO4" from phosphorylation (Figure 4). 

Specific ester substrates are also hydrolyzed with carboxypeptidase A. For instance, Makinen, Fukuyama, and Kuo (27) have recently studied the enzymic hydrolysis of 0-(trans-p-ch1orocinnamoyl)-L-B-phenyl actate (CICPI.) (47),and the spin labeled nitroxide ester substrate 0-3-(2,2,5,5-tetramethylpyrrol-inyl-l-oxyl)-propen-2-oyl-L-B-phenyllactate (TEPOPL) (48). They have shown that these reactions take place via the formation of a covalent intermediate (the mixed anhydride) which can be isolated under subzero temperature conditions. The hydrolysis of CICPL and TEPOPL catalyzed by carboxypeptidase A is consequently governed by the rate-limiting breaking of the acyl-enzyme. 

Conformer 50 has the proper electron pair orientation to break down to give either the free ester substrate and the enzyme (cf 49) or the mixed anhydride intermediate 52. The former process is however highly favored over, the latter because (a) a carboxylate group is a much better leaving group than an alkoxy group and (b) the electron pair of an alkoxy group (RO—) is... 

The X-ray structures of enzyme and enzyme-inhibitor complexes permit the anhydride intermediate only in the case of carboxypeptidase, not in the case of thermolysin, since in this enzyme the catalytic Glu-143 is too far away from the substrate carbonyl (Lipscomb, 1983). The proposal that carboxypeptidase works via an anhydride intermediate thus requires the supposition that two very similar enzymes work by different mechanisms. 

---